The type 3 secretion system requires actin polymerization to open translocon pores.

Many bacterial pathogens require a type 3 secretion system (T3SS) to establish a niche. Host contact activates bacterial T3SS assembly of a translocon pore in the host plasma membrane. Following pore formation, the T3SS docks onto the translocon pore. Docking establishes a continuous passage that enables the translocation of virulence proteins, effectors, into the host cytosol. Here we investigate the contribution of actin polymerization to T3SS-mediated translocation. Using the T3SS model organism Shigella flexneri, we show that actin polymerization is required for assembling the translocon pore in an open conformation, thereby enabling effector translocation. Opening of the pore channel is associated with a conformational change to the pore, which is dependent upon actin polymerization and a coiled-coil domain in the pore protein IpaC. Analysis of an IpaC mutant that is defective in ruffle formation shows that actin polymerization-dependent pore opening is distinct from the previously described actin polymerization-dependent ruffles that are required for bacterial internalization. Moreover, actin polymerization is not required for other pore functions, including docking or pore protein insertion into the plasma membrane. Thus, activation of the T3SS is a multilayered process in which host signals are sensed by the translocon pore leading to the activation of effector translocation.

Coronavirus Disease 2019 in a Patient With a Systemic Autoinflammatory Syndrome due to an NLRC4 Inflammasomopathy.

The effect of autoinflammatory diseases on severe acute respiratory syndrome coronavirus 2 infection remains unknown. We report a case of coronavirus disease 2019 (COVID-19) in a patient with autoinflammation with infantile enterocolitis with inflammatory flares due to a mutation in the inflammasome component NLRC4. This case highlights the role of immunosuppression in patients with autoinflammation with COVID-19.

Topology and Contribution to the Pore Channel Lining of Plasma Membrane-Embedded Shigella flexneri Type 3 Secretion Translocase IpaB.

Shigella spp. are human bacterial pathogens that cause bacillary dysentery. Virulence depends on a type 3 secretion system (T3SS), a highly conserved structure present in multiple important human and plant pathogens. Upon host cell contact, the T3SS translocon is delivered to the host membrane, facilitates bacterial docking to the membrane, and enables delivery of effector proteins into the host cytosol. The Shigella translocon is composed of two proteins, IpaB and IpaC, which together form this multimeric structure within host plasma membranes. Upon interaction of IpaC with host intermediate filaments, the translocon undergoes a conformational change that allows for bacterial docking onto the translocon and, together with host actin polymerization, enables subsequent effector translocation through the translocon pore. To generate additional insights into the translocon, we mapped the topology of IpaB in plasma membrane-embedded pores using cysteine substitution mutagenesis coupled with site-directed labeling and proximity-enabled cross-linking by membrane-permeant sulfhydryl reactants. We demonstrate that IpaB function is dependent on posttranslational modification by a plasmid-encoded acyl carrier protein. We show that the first transmembrane domain of IpaB lines the interior of the translocon pore channel such that the IpaB portion of the channel forms a funnel-like shape leading into the host cytosol. In addition, we identify regions of IpaB within its cytosolic domain that protrude into and are closely associated with the pore channel. Taken together, these results provide a framework for how IpaB is arranged within translocons natively delivered by Shigella during infection. IMPORTANCE Type 3 secretion systems are nanomachines employed by many bacteria, including Shigella, which deliver into human cells bacterial virulence proteins that alter cellular function in ways that promote infection. Delivery of Shigella virulence proteins occurs through a pore formed in human cell membranes by the IpaB and IpaC proteins. Here, we define how IpaB contributes to the formation of pores natively delivered into human cell membranes by Shigella flexneri. We show that a specific domain of IpaB (transmembrane domain 1) lines much of the pore channel and that portions of IpaB that lie in the inside of the human cell loop back into and/or are closely associated with the pore channel. These findings provide new insights into the organization and function of the pore in serving as the conduit for delivery of virulence proteins into human cells during Shigella infection.

Shigella flexneri Disruption of Cellular Tension Promotes Intercellular Spread.

During infection, some bacterial pathogens invade the eukaryotic cytosol and spread between cells of an epithelial monolayer. Intercellular spread occurs when these pathogens push against the plasma membrane, forming protrusions that are engulfed by adjacent cells. Here, we show that IpaC, a Shigella flexneri type 3 secretion system protein, binds the host cell-adhesion protein ß-catenin and facilitates efficient protrusion formation. S. flexneri producing a point mutant of IpaC that cannot interact with ß-catenin is defective in protrusion formation and spread. Spread is restored by chemical reduction of intercellular tension or genetic depletion of ß-catenin, and the magnitude of the protrusion defect correlates with membrane tension, indicating that IpaC reduces membrane tension, which facilitates protrusion formation. IpaC stabilizes adherens junctions and does not alter ß-catenin localization at the membrane. Thus, Shigella, like other bacterial pathogens, reduces intercellular tension to efficiently spread between cells.